利用抗膜結合免疫球蛋白亞型特異性片段單株抗體調控人類B細胞

Manipulating Human B cells with Anti-migis Monoclonal Antibodies

陳念宜(CHEN, NIEN YI)

指導教授 ： 張子文 ； 張晃猷

國立清華大學/生命科學院/分子醫學研究所/博士(2014年)

https://doi.org/10.6843/NTHU.2014.00183

摘要

在B細胞發育成熟的過程中，細胞膜上表現各種亞型的膜結合免疫球蛋白(亦稱為B細胞受體)，膜結合免疫球蛋白透過本身以及其他輔助受體，調控B細胞的增殖或凋亡。膜結合免疫球蛋白特殊之處，在於接近細胞膜的位置有一段序列專一性的表現於膜結合免疫球蛋白，稱之為膜結合免疫球蛋白亞型特異性片段(migis)，而且不同亞型的免疫球蛋白擁有獨特的膜結合免疫球蛋白亞型特異性片段。這種特性提供了發展調控不同亞型抗體的方法，發展膜結合免疫球蛋白亞型特異性片段專一性抗體，便能調節該類型抗體的B細胞，而且不受血液中游離抗體的中和作用干擾。我們針對IgM與IgD兩種亞型進行研究，利用融合瘤細胞技術，我們開發了一系列的單株抗體，專一性的結合至膜結合免疫球蛋白亞型特異性片段，這些抗體不僅專一性的辨識細胞上的migis序列，透過二次抗體的交叉連結，能誘導細胞凋亡以及引發抗體依賴型細胞毒殺反應。小鼠異體移植人類B細胞的動物模型中，給予抗 migis抗體能減少B細胞族群，並且降低體內IgM的濃度。由於抗migis抗體結合B細胞強度不若抗Fc抗體，推測是受到B細胞受體複合物的立體障礙，B細胞使用膽固醇提取試劑處理後，破壞B細胞受體複合物結構，顯著提高抗抗migis抗體結合B細胞強度。我們也利用抗體Fab片段與migis胜肽共同結晶，探尋抗體與migis結合關鍵位置，並解析抗膜結合免疫球蛋白亞型特異性片段的立體結構。這些結果證實以抗體標的膜結合免疫球蛋白亞型特異性片段來調節人體B細胞是可行的策略，目前開發的抗體雖然效價未臻完美，但透過對於結合免疫球蛋白亞型特異性片段立體結構的認識，將有助於開發出效果更好的抗體，使透過抗體標的膜結合免疫球蛋白亞型特異性片段以調節人體B細胞的策略，更接近實用價值。

關鍵字

B細胞 ； 單株抗體 ； 免疫球蛋白 ； B細胞受體

並列摘要

During the development and differentiate of B cell, various isotypes of membrane bound immunoglobulin (mIg) were expressed on the B cell surface. Membrane bound immunoglobulin itself and co-receptors deliver the proliferation and apoptotic signals upon crosslinked by antigens to regulate the B cell development. The heavy chain of mIg differs from that of secreted immunoglobulin in that mIg contains a C-terminal membrane-anchor peptide. Our group previously proposed that the N-terminal extracellular segment of the membrane-anchor peptide of mIg, referred to as the mIg isotype-specific segment (migis), may provide an unique antigenic site for isotype-specific targeting of mIg+ B cells. Here we report the preparation of mouse mAbs specific for human migis-delta and migis-mu. The mAbs bound to human migis containing recombinant proteins in an ELISA, to mIg-expressing transfectants of a CHO cell line and human B cell lines as analyzed by flow cytometry. MC116 cell, which is a mIgD and mIgM expressing human B cell line, could be induced to undergo apoptosis by treatment with those mabs in the presence of a second crosslinking antibody. The production of human IgM by transplanted MC116 cells in NOD-SCID mice could be suppressed by treating with the anti-migis-delta antibody, 20E6. The structure of migis was studied by co-crystallization of anti-migis Fab and migis peptide. These results encourage further investigation of the potential of anti-migis mAbs to control mIg+ B cells, when such a manipulation may alleviate a disease state.

並列關鍵字

B cell receptor ； membrane bound immunoglobulin ； monoclonal antibody ； migis

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